>what I want to find out are the
>optimum d:dd ratios for the 4 nucleotides.
>>I would also appreciate any data on thermal stability of ddNTPs. Is it
>because they get degraded that one has to use such high concs ? Or is it
>simply because the traditional "labeling reaction" is running in parallel
>with the "termination reaction"?
I've not yet tried cycle sequencing, though I hope to start soon, but my
experience with Klenow probably applies. I found that I had to adjust the
dNTP/ddNTP ratios (yes, by trial-and-error) from those given in protocols
in the literature to optimize things for my sequence. (One of the reasons
I went to Sequenase when it was available.)
You might want to look at BRL's FOCUS vol 5 #1 (March 1983) for trouble-
shooting regular dideoxy sequencing.
(ddNTPs should be stable.)
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